TGFβ-dependent expression of PD-1 and PD-L1 controls CD8+ T cell anergy in transplant tolerance

RESEARCH ARTICLE TGFb-dependent expression of PD-1 and PD-L1 controls CD8+ T cell anergy in transplant tolerance Marije Baas1,2,3†‡, Alix Besançon1,2,3†, Tania Goncalves1,2,3, Fabrice Valette1,2,3, Hideo Yagita4, Birgit Sawitzki5, Hans-Dieter Volk5,6, Emmanuelle Waeckel-Enée1,2,3, Benedita Rocha1,7, Lucienne Chatenoud1,2,3, Sylvaine You1,2,3* 1 University Paris Descartes, Sorbonne Paris Cité, Paris, France; 2Institut National de la Santé et de la Recherche Médicale Unit 1151, Institut Necker-Enfants Malades, Paris, France; 3Centre National de la Recherche Scientifique UMR 8253, Institut Necker-Enfants Malades, Paris, France; 4Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; 5Institute of Medical Immunology, Charité University Medicine, Berlin, Germany; 6Berlin-Brandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany; 7Lymphocyte Population Biology Unit, Pasteur Institute, Paris, France Abstract CD8+ T cell anergy is a critical mechanism of peripheral tolerance, poorly investigated *For correspondence: sylvaine. † These authors contributed equally to this work Present address: ‡Department of Nephrology, Radboud University Medical center, Nijmegen, The Netherlands Competing interests: The authors declare that no competing interests exist. in response to immunotherapy. Here, using a pancreatic islet allograft model and CD3 antibody therapy, we showed, by single cell gene profiling, that intragraft CD8+ lymphocytes coexpressing granzyme B and perforin were selectively depleted through the Fas/FasL pathway. This step led to long-standing anergy of the remaining CD8+ T cells marked by the absence of cytotoxic/ inflammatory gene expression also confirmed by transcriptome analysis. This sustained unresponsiveness required the presence of the alloantigens. Furthermore, tissue-resident CD8+ lymphocytes produced TGFb and expressed the inhibitory receptors PD-1 and PD-L1. Blockade of TGFb downregulated PD-1 and PD-L1 expression and precipitated graft rejection. Neutralizing PD1, PD-L1 or TGFbRII signaling in T cells also abrogated CD3 antibody-induced tolerance. These studies unravel novel mechanisms underlying CD8+ T cell anergy and reveal a cell intrinsic regulatory link between the TGFb and the PD-1/PD-L1 pathways. DOI: 10.7554/eLife.08133.001 Funding: See page 16 Received: 15 April 2015 Accepted: 02 January 2016 Published: 29 January 2016 Reviewing editor: Satyajit Rath, National Institute of Immunology, India Copyright Baas et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Introduction Upon antigen recognition, CD8+ T lymphocytes proliferate vigorously and differentiate into effector cells characterized by their ability to produce cytokines and chemokines, to migrate to inflamed tissues and to use various cytolytic pathways to kill their targets (Williams and Bevan, 2007). CD8+ T cells play major role in transplant rejection. Through direct presentation, they get activated by donor antigen-presenting cells (APC), in particular dendritic cells, which migrate from the graft to secondary lymphoid organs (Gras et al., 2011; Ochando et al., 2006). This response is rapid, intense and induces acute graft rejection. Recipient APC also capture donor antigens from the transplant, present them to alloreactive CD8+ T cells through self MHC I molecules (cross-priming/indirect presentation) hereby contributing to acute and chronic graft rejection (Celli et al., 2011; Valujskikh et al., 2002). Cytotoxic CD8+ T lymphocytes migrate to the graft where they destroy target cells through granzyme B/perforin- and/or Fas/FasLigand-dependent cytolytic pathways. Baas et al. eLife 2016;5:e08133. DOI: 10.7554/eLife.08133 1 of 19 Research article Immunology eLife digest The immune system is always on guard for signs of infection or cells that have become diseased. When these signs are identified, a subset of white blood cells called CD8+ T cells leap into action, multiply in number and then act to eliminate the potential threat. While this response is essential to fighting off infections and other diseases like cancer, it can backfire in people with an organ transplant. Indeed, the CD8+ T cells can target and attack the cells of the transplanted organ causing the body to reject the organ. One way to avoid transplant rejection would be to turn off CD8+ T cells that have learned to recognize cells from the transplant. In fact, studies in 2012 and 2013 showed that treating transplanted animals with an antibody that binds T cells protects a transplanted organ from attack. This treatment had to be given after the CD8+ T cells had recognized and began targeting the transplanted organ to be effective. But it was not clear exactly how this antibody treatment protected the transplant. Now, Baas, Besançon et al. – including some of the same researchers involved in the earlier studies – show that the antibodies used in the treatment selectively target and eliminate the attacking CD8+ T cells. This leaves behind only inactive CD8+ T cells that don’t harm the transplant. To do this, Baas, Besançon et al. transplanted pancreatic cells from mice into other mice with a diabetes-like disorder. Next, the experiments compared gene expression in CD8+ T cells found within the transplanted tissue in mice that were treated with the antibody and those that were not treated. The expression of many genes for toxic molecules was stopped after treatment with the antibody leaving the CD8+ T cells in an inactive state. In addition, the treated CD8+ T cells expressed more of a certain type of receptor (called PD-1 and PD-L1) that acts as inhibitory checkpoint for the immune system. So, Baas, Besançon et al. treated transplanted mice with both the T cell-eliminating antibody and antibodies that block these inhibitory receptors to see what would happen. The transplanted organs were quickly attacked and rejected. This shows that the inhibitory receptors play a crucial role in helping to shut down attacking CD8+ T cells in the initial antibody treatment and allowed long-term survival of the transplanted organs. Blocking another protein called TGFb in antibody-treated mice also caused organ rejection. The findings help explain how these antibodies protect transplanted organs and may help scientists trying to develop new anti-transplant rejection drugs in the future. DOI: 10.7554/eLife.08133.002 In most tolerance promoting protocols, long-term graft survival was associated with CD8+ T cell dysfunction which mainly resulted from clonal deletion and/or anergy (Iwakoshi et al., 2000; Monk et al., 2003; Qian et al., 1997). Classically defined as the functional inactivation of T cells to cognate antigens, anergy was first described in vitro when T cells recognized antigens (signal 1) in absence of app (...truncated)